Bruce H. Sells

Modification of ribosomal proteins during liver regeneration.

Abstract Analysis of the 2D gel electrophoretic pattern of ribosomal proteins from both the small and large subunit of rat liver were made at various times following partial hepatectomy. No changes were observed in the electrophoretic mobility of proteins from the 60S subunit during periods of 2 hr. to 72 hr. of liver regeneration. Changes were observed, however, in two proteins of the 40S subunit a short time after partial hepatectomy. Protein S6 disappeared from its normal position and a new spot appeared as a more negative form as early as 2 hr. post regeneration. This modification persisted for at least 18 hr. At 72 hr., S6 returned to its normal position. Protein S2, on the other hand, underwent a different pattern of change during the early stages of liver regeneration. S2 was observed to migrate as 2 spots at 2 hr. after partial hepatectomy and this pattern was preserved at the 4 hr. period. At 8 hr., the pattern was further modified to 2 spots which was distinct from the earlier change. This pattern was similar at 12 hr. At 18 hr. only the normal S2 protein was observed. No further change in S2 migration was observed at the 72 hr. period of liver regeneration.

Cytoplasmic nonpolysomal ribonucleoprotein complexes and translational control

SummaryIn this article, we discuss our attempts to establish the existence in the cytoplasm of regulatory molecules involved in translational control. Our studies have revealed the presence of cAMP independent protein kinase in the free mRNP complex capable of phosphorylating a Mr = 38 000 polypeptide, also part of the same complex. Both the kinase and the acceptor protein were found also as free proteins in the cytoplasmic pool. This kinase has been shown to be distinct from the heme regulated enzyme that phosphorylates the small subunit of eIF-2.Other regulatory molecules include small molecular weight RNAs found as part of an RNP complex. A 4S fraction isolated from this complex inhibited the translation of both capped and uncapped mRNAs in a cell-free protein synthesizing system.The biological role of the protein kinase and the 4S RNA fraction is considered.

Molecular morphology of ribosomes: Effect of chain initiation factor 3 on 30 S subunit conformation

Chain initiation factor 3 (IF-3) binds to a single site on the Escherichia coli 30 S ribosomal subunit [l-4] . Recent studies have indicated that this binding site is in close proximity to a number of ribosomal proteins [4-81. Various lines of evidence suggest that the association of the factor with 30 S subunits causes a conformational change of the particles [9,10] . Such a change may be responsible for the activity of IF-3 in promoting the binding of message to ribosomes [ 1 l141, and in preventing ribosomal subunit association [15-l 81. However, the specific components of the 30 S subunit which are involved in the conformational change induced by IF-3 are not known. For the work presented here, lactoperoxidasecatalyzed iodination was used to examine the influence of IF-3 upon the conformation of the 30 S ribosomal subunits. This probe has previously allowed us to demonstrate structural alterations of ribosomes following changes in ionic strength, temperature and selective removal of proteins L7 and L12 from the 50 S subunit [ 19-221. Our studies reveal that the enzymatic iodination of 5 ribosomal proteins is altered following the 30S-IF-3 interaction. 2. Materials and methods

Comparison of the temperature sensitivity of protein synthesis by cell-free systems from liver of rat and skate (Raja ocellata

Studies were undertaken to determine the component(s) responsible for the temperature optimum characteristic of the protein-synthesizing system from skate and rat. 1. The macromolecular constituents of rat and skate liver ribosomes are compared. The number of ribosomal proteins is similar in the two species, although most proteins display different electrophoretic mobilities on polyacrylamide gels. The RNAs from the small subunit of skate and rat have similar sedimentation coefficients; however, the RNA from the large subunit of skate ribosomes appeared to be slight smaller than the comparable RNA from the rat. 2. Ribosomes from either rat or skate were capable of supporting poly(U)dependent polyphenylalanine synthesis with soluble factors from either species. 3. Maximal leucine incorporation directed by endogenous mRNA occurred at 35--40 degrees C with post-mitochondrial supernatant from the rat liver and at 20--30 degrees C with that from skate liver. 4. The characteristic temperature sensitivity of protein synthesis was dependent upon the source of cell sap and independent of the source of ribosomes. 5. Elongation factor 1 from both the rat and skate exhibited maximum activity at approx. 30 degrees C. 6. Phenylalanyl-tRNA synthetase from skate liver showed maximum activity at 30 degrees C while that from rat was maximally active at 37 degrees C. The rat enzyme, however, was active at 0--10 degrees C, at which temperature protein synthesis in the reconstructed rat system is virtually absent. 7. The protein-synthesizing capacity of the reconstituted system at various temperatures was closely correlated with the activity of Elongation factor 2 (translocase). Elongation factor 2 from rat liver displayed an optimum at 30 degrees C and lost all activity below 10 degrees C, while this same factor from skate liver showed an optimum at 20 degrees C and significant activity below 10 degrees C. At this low temperature the reconstituted skate liver system continued to exhibit the ability to synthesize protein. These studies suggest that Elongation factor 2 is the component responsible for determining the temperature at which the protein-synthesizing system displays its characteristic maximum activity.

Molecular morphology of ribosomes: Structural alteration of 50S subunits following the removal of proteins L7 and L12

Ribosomal proteins L7 and L12 are involved in the initiation, elongation and termination of polypeptide synthesis [l-6]. These acidic proteins do not function independently, but act in concert with the ribosome to perform the above steps. These observations suggest that L7 and L12 are necessary to maintain the conformational integrity of the ribosome, and that loss of ribosomal activity following the removal of L7 and L12 results from a structural alteration of the large subunit. The present investigation is an attempt to determine whether L7 and L12 are involved in preserving the structural conformation of the 50s subunit. Lactoperoxidase-catalyzed iodination of ribosomal proteins has been utilized as a probe to study ribosome structure [7-lo]. Although this probe was originally designed to investigate the surface topography of ribosomes, it has recently been employed to detect conformational alterations of ribosomal subunits accompanying changes in ionic strength, temperature and isolation conditions [ 1 1 -131. To determine whether structural alterations of the 50s subunit occur following the removal of L7 and L12 from the ribosome, we have again used the lactoperoxidase-catalyzed iodination system. Investiga. tions have been designed to detect changes in reactivity of individual ribosomal proteins following the removal and readdition of L7 and L12 as measured by enzymic iodination. 2. Materials and methods

Polyamine levels in Escherichia coli during nutritional shiftup and exponential growth

At different exponential growth rates obtained either by varying the carbon source of the culture medium or limiting glucose uptake, intracellular levels of putrescine and spermidine were measured. Over a ten-fold increase in growth rate an approximately three-fold increase in putrescine level and a 3.5-fold increase in spermidine level per cell absorbance were observed. Conditions favoring an abrupt alteration in growth rate, such as occur following nutritional shiftup of Escherichia coli, resulted in a significant increase in the intracellular level of putrescine and virtually no change in the spermidine level. Because of the magnitude and the timing of the change in polyamine levels, the hypothesis that polyamines are (the components) responsible for inducing the rapid increase in the rate of RNA synthesis following nutritional shiftup is rejected.

The relationships among RNA synthesis, RNA polymerase synthesis and guanosine tetraphosphate levels in Escherichia coli during nutritional shift-up

Abstract The relationships among the rate of RNA synthesis, RNA polymerase synthesis and activity, and guanosine tetraphosphate levels were investigated following nutritional shift-up in Escherichia coli. RNA synthesis continues at the preshift rate for 1.5 min after which an increase is observed that reaches a new steady-state rate at between 2 and 2.5 min. RNA polymerase activity measured in crude extracts increases immediately and by 10 min has increased 50%. RNA polymerase synthesis as measured by the synthesis of the β and β′ subunits lags for 2.5 min and then increases 75% by 10 min. Guanosine tetraphosphate levels decrease 50% by 3 min to levels characteristic of steady-state post-shift-up cells. The significance of these data to the regulation of RNA synthesis during shift-up is discussed.

Activity of polypeptide chain initiation factors during a nutritional shift-up in Escherichiacoli

Summary During the first 10–15 minutes following nutritional shift-up in Escherichia coli , no significant increase was detected in the specific activity of initiation factors. The next 25 minutes, however, were characterized by a doubling of specific activity of these factors. Measurements of initiation factor activity of different exponential growth rates revealed a linear proportionality between growth rate and initiation factor activity. For each doubling of growth rate, initiation factor activity increased 1.6–1.8 fold.

The effect of magnesium starvation on the dissociation of ribosomal proteins from Escherichia coli K-12 ribosomes

The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli. During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit. An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus [5] as tightly bound. Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+. These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits.

Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins of Eschrichia coli

SummaryThe rates of functional decay of messenger RNA coding for total soluble, total ribosomal and individual ribosomal proteins were measured in Escherichia coli strain AS-19, at 30o. This was accomplished by blocking RNA synthesis with the inhibitor thiolutin and measuring residual protein synthesis at various times thereafter. The data obtained expressed as a decay constant (Hartwell and Magasanik, 1963) show that both total soluble and total ribosomal protein decay with similar rates (K2=0.64 and 0.61 respectively) which are slightly faster than the decay rate of β-galactosidse (k2=0.43) under these conditions. All the individual ribosomal proteins appear to comprise a population of cistrons whose individual mRNAs decay with very similar rates with the possible exception of protein L3, whose mRNA appears consistently to decay very rapidly.Additional data on the stability of the total soluble and total ribosomal proteins during thiolutin treatment (that is, proteins synthesized in the absence of concommitant ribosomal RNA synthesis) fail to demonstrate any marked difference between these two protein populations. Examination of the stability of the individual ribosomal proteins however, reveals that some are degraded up to 35% in 15 min of thiolutin exposure, some to about 15% and some appear to be completely stable. In general, a degree of correlation exists between the stability of a given protein and the observed decay rate of its messenger RNA. This observation may explain in part the spread among the rates of mRNA decay. Nevertheless, we conclude that although degradation is occurring, it is not sufficient to alter the main conclusion that the rates of functional decay of mRNA cistrons coding for the ribosomal proteins are very similar.